1. Field of the Invention
The invention relates to the methods to manufacture through fractionation and purification of human albumin, immunoglobulins, clotting Factor VIII and clotting Factor IX, from human plasma by sequential chromatography steps to produce highly pure, virus-free and therapeutically administrable proteins. The purification is carried out by an all-chromatography process that avoids the use of ethanol precipitation.
2. Background Art
The first reported plasma fractionation process for therapeutic purposes was developed about 60 years ago by Cohn and co-workers (J. Am. Chem. Soc., 68, 459-475, 1946; J. Am. Soc. 72, 465-474, 1950). Since then, plasma fractionation industry has grown mani-fold and is now one of the largest industry segments in global therapeutic protein manufacture (J. Curling, Integrating new technology into blood plasma fractionation BioPharm September 2002, 16-25; Adil Denizli, Plasma Fractionation: conventional and chromatographic methods for albumin purification J. Biol. Chem., 39(4), 315, 341, 2011).
Plasma protein fractionation is approximately an 11.8 billion dollar industry (Plasma Product Biotechnology meet 2011, Dr. Jean-Francois Prost, “Will plasma products inevitably be replaced by a new generation of therapeutics?”) supplying products to more than a million patients each year. It is estimated that more than 500 metric tons of human serum albumin and more than 40 tons of intravenous immunoglobulin are produced annually from more than 22 million liters of source and recovered plasma. Plasma contains about 60 g/L of protein, of which about 57 grams (not including processing losses) are used for many therapeutic products. Plasma-derived products, such as IVIG and albumin, are not expected to be manufactured by recombinant means. IVIG demand is now the primary driver of the plasma collection market, with demand growing 6-8% annually.
According to a market report published by Robert, P. in Pharmaceuticals Policy and Law 11, 359-367, 2009, the demand for plasma in the 1950s and 60s was driven by the demand for human albumin. In the late 1960s, when Factor VIII concentrates became available, the global plasma requirement was driven by the growing demand for clotting factors. The IVIG usage has grown significantly in different medical areas such as neurology, rheumatology, nephrology, dermatology, oncology and infectious diseases, as well as allergy and immunology, in addition to the continuing increase of the number of patients with primary immune deficiencies. Based on this usage pattern, which characterizes the markets in most industrialized countries, the volume of IVIG was forecast to grow from about 82.3 metric tons in 2008 to about 107.9 tons by 2012, corresponding to an annual growth rate of 7%—the rate observed in the past ten years. According to this report, the demand of IVIG beyond 2012, is expected to grow depending upon the results of the Alzheimer's disease trials and the possible approval of IVIG for this new indication.
In 2010, there were a total of 78 fractionation plants operating in the world, with 25 of them in China, 26 in Europe and 8 in United States (Worldwide supply and demand of plasma and plasma-derived medicines, Patrick Robert, Iranian J. of Blood and Cancer, Vol. 3, No. 3, 111-116, 2011). The global fractionation capacity was estimated at about 48.4 million liters, producing mainly albumin, polyvalent IVIG, Factor VIII, Factor IX and Hyperimmunes. Hemophiliacs are the target population for players interested in plasma protein R&D. India needs about 900,000 liters of plasma derived proteins per year. India has been meeting this requirement so far by importing these proteins as the plasma fractionation industry is still at a nascent stage, one of the major hindrances being the existence of adequate infrastructure for plasma collection (Farrugia A., Plasma for Fractionation: Safety and Quality Issues. Haemophilia 10, 2004: 334-340).
Although the number of plasma fractionators are growing and global capacities are increasing, so is the demand for IVIG growing, which is projected at about 7% to 13% annually between 2012 and 2015. To meet this demand, more raw plasma will need to be dedicated to immunoglobulin purification, along with improvements in the process that will increase the overall IVIG yield. This growing requirement for IVIG will limit the availability of plasma for the manufacture of new plasma-derived blood products unless their manufacture is integrated into the existing manufacturing processes for plasma-derived products such as immunoglobulins and albumin. Presently, although plasma based therapeutics are safe, efficacious and available, the cost of therapy remains a barrier for access to the drugs. This can be addressed only if human plasma, a uniquely rich starting material for therapeutic proteins, can be processed to prepare several more therapeutic proteins besides the conventional albumin, IVIG and clotting factor concentrates. This also requires the use of more discerning technologies of bioprocess operations like chromatography and membrane separations together with the most recent and proven methods of viral inactivation.
Plasma being a scarce and exclusive commodity, there is a continuous need to upgrade the fractionation processes so as to maximally utilize this valuable resource. By continuously refining and upgrading the existing processes and improving process efficiencies, the market availability status and the quality of the end products can be improved. The fractionation industry which presently uses either a Cohn or a modified Cohn's method of ethanol precipitation in some cases may use chromatography based purification in the subsequent steps. Cohn's fractionation limits the number of proteins that can be purified by ethanol precipitation as only the more abundant proteins from plasma can be precipitated by this method. The less abundant therapeutic proteins with good therapeutic potential need to be processed using a different strategy. It's also now accepted in the plasma fractionation industry that although Cohn's method permits fractionation of large volumes of plasma at low cost, the quality of the product obtained by chromatography is superior (Braz. J. Med. Biol. Res., 31:1383-1388, 1998). Several companies have now moved to usage of a combination of both methods, where they have ethanol precipitation followed by chromatography. The inherent disadvantage in this method is the possibility of protein denaturation and/or aggregation during the addition of ethanol (Braz. J Med Biol Res, 31: 1375-1381, 1998). Protein losses may also occur in the supernatant after ethanol precipitation thereby decreasing the overall recovery of major proteins and almost a total loss of the minor proteins. The concentration of different proteins in the plasma varies over a very wide range, from less than 1 microgram per ml to 40 grams per liter. To develop separation and purification processes that can ensure production of several of these high and low level proteins from the same starting volume of plasma, is a challenge for the separation specialists.
There are several patents in the prior art that disclose a process for fractionation and/or purification of plasma proteins to therapeutic grade purity, that involved the use of ethanol for initial precipitation, followed by chromatography steps with few technologies involving only chromatographic steps or only ethanol precipitation steps.
Preparation of a high-purity human Factor IX concentrate and other plasma proteins and their therapeutic use with chromatography process were discussed in U.S. Pat. No. 5,457,181. U.S. Pat. No. 4,371,520 discloses a process for preparing immunoglobulin suitable for intravenous injection, comprising treatment of an acid a plasma or combination of fractions I, II and III, combination of fractions II and III, fraction II, or fraction III obtained from plasma by Cohn's cold alcohol fractionation and chromatography methods. Process for the isolation and/or fractionation of peptide, 5 polypeptides or protein solutions discussed in WO200763129, involves both chromatographic steps and ethanol precipitation steps. Patent application WO200623831 relates to chromatographic methods for recovering highly purified proteins sequentially from biological samples. U.S. Pat. No. 5,679,776 discloses a simple chromatographic process for purifying Factor VIII from total plasma. However, none of the above referred patents discloses a process that can simultaneously isolate and purify several proteins from the same starting volume from human plasma to therapeutic grade proteins by avoiding the use of ethanol precipitation step anywhere in the process.
Another patent, U.S. Pat. No. 7,041,798 discloses a method for the chromatographic fractionation and ethanol precipitation of plasma and serum preparations, and relates to the fractionation of plasma or serum into albumin and immunoglobulin by hydrophobic interaction chromatography. The rate limiting step is the processing time associated with starting material which can be challenging at large scale.
Patent U.S. Pat. No. 4,639,513 discloses a method for producing intravenously injectable immunoglobulin G (IgG) comprising a particulate separation step, an ion exchange separation step and an affinity separation step, ethanol precipitation, additionally useful high purity by-products such as prothrombin complex, transferrin and albumin recovery, were published. However, these methods often employ a separation medium that can be designed to selectively adhere either the protein of interest or the impurities.
Patents U.S. Pat. No. 4,877,866 and DE 3640513C2 disclose the methods appropriate for industrial-scale production and multistage purification of plasma that contains immunoglobulin G accompanied by treatment with ultrafiltration and ethanol precipitation. However, a need exists for an efficient process for purifying an immune serum globulin fraction from a crude plasma protein fraction.
The patent application WO1994029334 discloses chromatographic and ethanol precipitation steps to produce therapeutic quality of the IVIG. However, the claimed method fails to take the subsequent purification of albumin into account.
Although a number of chromatography based processes have been described in research publications for the production of IVIG without the use of ethanol precipitation, but at the industrial scale, an all-chromatography process scheme has just begun gaining acceptance due to the better quality of the final IVIG product (lesser protein denaturation and aggregation) and better yields (Lontos, J., Chromatographic purification of immunoglobulins at CSL bioplasma; a manufacturing perspective. Plasma Product Biotech meet, www.bo-conf.com/ppb05/present/ppt.htm 2005; Bertolini, J., Davies, J., Wu, J., Coppola, G., Purification of Immunoglobulins. 1998, WO 98/05686; Ultra-high yield intravenous immune globulin preparation, Zurlo, E. J., Curtin, D. D., Louderback, A. L., US patent application US20070049733A1).
Several patents have explored the use of chromatographic methods for the final purification steps particularly ion exchange chromatography. A few patents have also tried to replace the ethanol fractionation of plasma with caprylate or citrate precipitation steps followed by chromatography based clean-up steps for purification. In spite of these advancements, it is glaringly apparent how Cohn's fractions II and III continue to play an important role as the starting material for IVIG purification in most industrial processes. This is clearly evident in a patent being granted (U.S. Pat. No. 6,893,639 B2) to an ethanol based plasma fractionation process at sub-zero temperatures, as recent as 2005 (the initial Cohn's patent for ethanol based plasma fractionation U.S. Pat. No. 2,390,074, was granted in 1945). Cohn himself had illustrated the problems of protein denaturation as an undesirable outcome of ethanol precipitation.
Similar issues are applicable for human albumin as well. The industrial processes for purifying albumin start with Fraction V of ethanol fractionation of plasma by the Cohn's method (J. Am. Chem. Soc. 1946, 68, 459-475, Cohn et al.). Although ethanol fraction supernatants of fraction IV and even supernatants of fractions II & Ill have been used by several manufacturers for albumin purification, followed by chromatography steps, a complete all-chromatography process has not been the norm in the industry for albumin purification (U.S. Pat. No. 5,346,992 and U.S. Pat. No. 4,288,154). Chromatography techniques for albumin purification in research publications and books have been cited since the 1980s (Curling J. M., Methods of plasma protein fractionation, Ed. J. Curling, Acad. Press, 77-91 (1980); Saint-Blancard J., Novel Trisacryl ion exchangers (Nouveaux echangeurs d′ions Trisacryl), Ann. Pharm. Fr. 39, 403-409 (1981)). Patents that claim to purify albumin by chromatography are also seen to use supernatant IV or precipitate V of the Cohn's fraction (U.S. Pat. No. 5,677,424; U.S. Pat. No. 4,675,384; U.S. Pat. No. 4,228,154; U.S. Pat. No. 8,088,416) as the starting material.
U.S. Pat. No. 5,061,789 discloses a method for isolating blood-clotting Factor IX by first adsorbing it onto a matrix derivatised with alpha hydroxylamine groups, eluting the factor and adsorbing it onto a column matrix of sulphated carbohydrates to collect a pure form of Factor IX at high yields. U.S. Pat. No. 5,138,034 discloses a process for isolation of the Factor IX by a sequence of steps involving ethanol precipitation, followed by treatment with an anion exchanger and affinity chromatography. U.S. Pat. No. 5,286,849 discloses the process for purification of Factor IX from an impure protein fraction by the addition of a solvent-detergent solution to inactivate any viral contaminants and purify the Factor IX by chromatography on a sulphated polysaccharide resin. The purified Factor IX by the above method is claimed to have a specific activity of at least 85 units/mg. U.S. Pat. No. 5,457,181 discloses a method for preparing a high purity Factor IX concentrate from the supernatant fraction of cryoprecipitated human plasma. A pre-purification is done with DEAE SEPHADEX® chromatography. The resulting Factor IX has a specific activity of at least 0.5 IU/mg of protein. The purification method comprises at least two successive chromatography steps. The first step is a DEAE SEPHAROSE® chromatography followed by an affinity on HEPARIN SEPHAROSE™. The elution buffer is a citrate buffer at pH 7.4 adjusted with 0.45M NaCl and supplemented with arginine as a stabiliser for Factor IX activity. U.S. Pat. No. 5,614,500 discloses a method for preparing pharmaceutical compositions comprising the active, highly purified and concentrated Factor IX proteins. The Factor IX proteins are recovered from plasma or recombinant cell culture sources by an immunoaffinity chromatography procedure in the presence of a chelating agent and in the absence of an exogenous non-chelating protease inhibitor. U.S. Pat. No. 5,639,857 discloses a method for recovering active, highly purified and concentrated Vit-K dependent proteins from plasma, concentrate or mixtures of proteins produced by recombinant DNA technology. U.S. Pat. No. 5,714,583 discloses a process for purification of Factor IX by following the steps of anion exchange resin, heparin or heparin like resin followed by a hydroxyl apatite resin. The third eluate can be applied to an immobilised metal-affinity resin to get a fourth eluate that contains purified Factor IX. This process would apply to plasma derived as well as recombinant Factor IX. U.S. Pat. No. 6,063,909 discloses a novel method of protecting blood coagulation Factor IX from proteases during purification or storage was disclosed. Factor IX is stabilized in solution against activation to Factor IX or against degradation to peptides or conformation by the addition of one or more soluble organic or inorganic salts like sodium sulphate, potassium chloride, sodium chloride and/or other salts in the range of 0.7M to 3M, to the Factor IX containing solution. U.S. Pat. No. 6,258,938 discloses a method to isolate the blood clotting Factor IX by using an antibody affinity column that specifically reacts with ligand stabilized protein to be isolated. The antibody to Factor IX is immobilized on to a solid support and a mixture containing the protein in the presence of the ligand is brought in contact with the antibody bound to the column resin. Elution is done under mild conditions to release the protein without the ligand. This gave a protein with a specific activity of at least 125-150 units/mg of protein. They claim that the stability of the purified Factor IX can be improved by the removal of or decreasing the amount of plasma hyaluronan binding protease that may get co-purified with Factor IX according to the invention described in US 20040106779A1. The invention describes the method to decrease the ratio of Factor IX to PHBP by contacting it with an ion exchange column and eluting the protein with 0.35M to 0.4M salt to separate protease from Factor IX.
EP617049 and U.S. Pat. No. 5,378,365 discloses a process for the purification of Factor IX, Factor X and Factor II from human plasma or fractions by chromatographic methods.
Alcohol precipitation and chromatography methods were used in U.S. Pat. No. 4,093,608, which have disclosed a process for separating coagulation Factor VIII from human fresh pooled plasma and purifying it. However, the process is expensive and also lacks specificity.
U.S. Pat. No. 5,245,014 discloses a method for isolating biological compounds such as proteins, especially coagulation Factor VIII in high yield and almost free of other proteins, from body chromatographic and alcohol precipitation methods. However, purity of the obtained Factor VIII seems to be a challenge.
U.S. Pat. No. 4,822,872 discloses a method of purifying an anti-hemophilic factor (AHF) comprising of a cryoprecipitation of Factor VIII and adsorption of the Factor VIII protein on a water insoluble carrier. But, this is a complex process involving the preparation of custom-made resins that is not easy for industrial scale manufacturing. Other patents that discuss isolation and/or purification processes include WO2007136327; US20010051708.
There was hence a requirement, to have an integrated sequential chromatography procedure using regular industrial chromatography resins, to manufacture several products from a given volume of plasma, which ensures maximum utilization of a scarce and precious resource like human plasma and thereby better revenues for the company manufacturing these products.
The present invention discloses an approach to overcome the challenge of utilization of a scarce and precious resource like human plasma, by using a sequential chromatography procedure that can help purify the four proteins Albumin, IgG, and Clotting factors (Factor VIII and Factor IX), with a potential to purify more proteins from the same starting volume of plasma. This will in turn reduce the cost and improve the affordability of these life-saving medicines for the patients.